1. Presented by Gordon Holt, Ph.D. at the Nonclinical Studies Subcommittee of the Advisory Committee for Pharmaceutical Science March 9, 2000

2. OGS Proteomics Technology: Overview

3. Challenges for Proteomics Validation ProblemSolution Sample variabilityProcess validation Low sensitivityImmunoaffinity enrichment Subcellular fractionation Fluorescent dyes Imaging Gel variabilityProcess validation Image warping Low throughputRobotics Data analysis overloadLIMS management

4. GenomicsProteomics Coverage per runAll genes2000 features Sensitivity limits 1 mol./cell100 mol./cell Protein modificationsNoYes Subcellular localizationNoYes Subunits / ComplexesNoYes Clinical samplesNoYes Comparison of Microexpression Analyses

5. Identification of Cardiotoxicity and Vasculitis Surrogate Markers Work done in collaboration with Frank Sistare CDER FDA

6. Investigation of Doxorubicin-Induced Toxicity Doxorubicin (Dxr) background commonly used anticancer agent –effective against childhood leukemia causes dose-related cardiotoxicity –precise toxicity mechanism unknown –metal ions appear to be important metal chelation by ICRF-187 provides significant chemoprotection >> Can proteomics identify clinically relevant early markers of cardiotoxicity?

7. Doxorubicin cardiotoxicity study design Three rats per group 1 mg/kg doxorubicin per week Treat for 7 weeks Sacrifice animals 24 hrs after last dose Proteomic analyses of plasma –OGS plasma sample SOP –serum enrichment applied

8. Detection of disease-specific proteins in serum, CSF, synovial fluid Limitation :High-abundance proteins limit sensitivity - albumin - haptoglobin - IgG - transferrin Solution :Immunoaffinity enrichment protocol

9. Normal Enriched

10. Rosetta TM Analyses Preliminary study performed on 18 PEMs –one PEM per plasma sample from each rat –approx. 1800 features in master group –32,400 features screened High-stringency marker selection criteria –98% marker confidence –100% marker incidence on PEMs from given group of three rats

11. Doxorubicin + ICRF vs. control Doxorubicin treatment markers -30 -20 -10 0 10 20 Fold Change 357925293134373940414243 26102122232426272830323335363844454647 Doxorubicin vs control

12. Summary of results 34 Dxr treatment markers identified –magnitude up to 28.1-fold >> may be clinically useful Dxr toxicity markers Most Dxr markers normalized by ICRF co- treatment –29 fully normalized –5 partially normalized >> supports ICRF’s protection against Dxr toxicity

13. Dxr Cardiotoxicity - Preliminary Annotations Lipid metabolism - liposome formation Immune surveillance - complement fixation Wound healing - scar formation - protease inhibition Anti-oxidant metabolism - metal scavenging

14. Vasculitis study design & status Three rats per group 100 mg/kg SKF 95654 Treat for 1, 2, 4, 24 hours Histology completed Proteomic analyses of plasma –OGS plasma sample SOP –serum enrichment applied –PEMs run –differential analysis underway

15. Identification of Nephrotoxicity Surrogate Markers Work done in collaboration with Quintiles UK

16. Gentamicin Background  Parenteral aminoglycoside active against gram- negative bacteria  Clinical important toxicity - potential for irreversible cumulative ototoxicity (manifest as hearing loss - initially of high frequencies) and vestibular damage  Reversible nephrotoxicity may occur and acute renal failure reported  Therapeutic index - individual monitoring of plasma concentrations generally required

17. 7 Days of treatment followed by 14 day recovery period Route of administration:intravenous Dose levels:0, 0.1, 1, 10, 40, 60 mg/kg/day Group size:10 male rats per treated group, 20 male rats in control group Blood and urine samples:2, 3 and 8 days Blood Parameters:BUN, creatinine Urine parameters:NAG, ALP, GGT, volume, specific gravity Renal histopathology:standard Proteome samples:420 Study Protocol

18.  Immunoaffinity enrichment  30 images  2,580 MCIs  21,001 features Serum Proteomics

19.  Single protein linked to regulation of alternate pathway of complement  Human proximal tubular epithelial cells specifically bind to components of the alternate complement pathway >> Appears at lower dose than identified by conventional means Summary to date

20. Identification of Breast Cancer Serum Surrogate Markers Work done in collaboration with Prof. C. Coombes CRC London

21. Breast Cancer - pilot serum marker study 17 normals 17 primary breast tumors 17 metastatic breast tumors >> Serum enrichment protocol

22. Breast cancer sera - Differentials feature change has p value 50% of PEMs 63 potential marker proteins identified Normal vs primary........16 diff. proteins Normal vs metastatic.....20 diff. proteins Primary vs metastatic....27 diff. proteins >> proteins consistent with breast disease stage >> new proteins previously unassociated with breast

23. Summary: OGS Proteomics Identify disease-specific proteins Identify treatment-specific proteins >> Quantitative and qualitative >> Synergy with genomics data >> Powerful tool for surrogate marker identification

24. Key Elements for Partnership to Identify Surrogate Markers Institutional mandates of major players –FDA / NIH –academic –industrial Development stage-specific pressures –discovery, validation, commercialization Intellectual property ownership –discoverers (probably) not developers